Process for recovering protein containing microbial cells

ABSTRACT

A process for recovering protein-containing cells which comprises culturing a microorganism selected from the group consisting of new species Corynebacterium fujiokense, Nocardia neoopaca, Arthrobacter rubellus and Arthrobacter pectroleophagus under aerobic conditions in a culture medium containing a gaseous C2-C4 hydrocarbon as a carbon source, and separating and recovering cells from the culture broth.

Iizuka et a1.

PROCESS FOR RECOVERING PROTEIN-CONTAINING MICROBIAL CELLS Inventors: Hiroshi lizuka; Naosuke Seto, both of Tokyo; Sadao Sakayanagi, Kawasaki, all of Japan Assignee: Nippon Oil Company, Ltd., Tokyo,

Japan Filed: Mar. 16, 1970 Appl. No.2 20,829

Foreign Application Priority Data 1451 Oct. 2, 1973 [56] References Cited UNITED STATES PATENTS 3,622,465 11/1971 Orgel et al. 195/28 R 3,384,491 5/1968 Guenter et al.... 195/28 X 3,414,477 12/1968 Douros et al 195/28 Primary Examiner-Joseph M. Golian AttorneySherman and Shalloway 57 ABSTRACT A process for recovering protein-containing cells which comprises culturing a microorganism selected from the group consisting of new species Corynebacterium fujiokense, Nocardia neoopaca, Arthrobacter rubeIIus and Arthrobacter pectroleophagus under aerobic conditions in a culture medium containing a gaseous C -C hydrocarbon as a carbon source, and separating and recovering cells from the culture broth.

8 Claims, No Drawings PROCESS FOR RECOVERING PROTEIN-CONTAINING MICROBIAL CELLS This invention relates to a process for recovering pro tein-containing microbial cells by culturing a new microorganism in a culture medium containing a gaseous hydrocarbon as its sole carbon source and recovering protein-containing cells from the culture broth. The process of the invention has many advantages over a process using liquid hydrocarbons as a carbon source and gives protein-containing cells havin an abundance of protein and vitamines at good yields.

More particularly, the present invention relates to a process for recovering protein-containing cells which comprises culturing a microorganism selected from the group consisting of Corynebacterium fujiokense, N- cardia neoopaca, Arthrobacter rubellus and Arthrobacter petroleophagus under aerobic conditions in a culture medium containing a gaseous C -C hydrocarbon as a carbon source, and separating and recovering cells from the culture broth.

In recent years, the fermentation industry has developed methods of fermentation wherein inexpensive and readily available hydrocarbons, such as natural gas, by-

product gases occurring in the cracking of petroleum,

naphtha and petroleum paraffins, are utilized as a carbon souce in place of the conventionally used carbohydrates, and various microorganisms which assimilate such carbon sources have been discovered and reported.

Microorganism cells which assimilate there hydrocarbons have attracted much attention because they are nutrient as protein sources for foodstuff and feeds for poultry, domestic animals, and fish, and can be produced on a commercial scale in great quantities for short periods of time to provide inexpensive protein sources.

I-Ieretofore, the research and development work in this field has been concentrated mainly on fermentations involving the use of liquid hydrocarbons such as n-paraffins as carbon sources, and there have been fewer reports made on fermentations involving gaseous hydrocarbons as carbon sources.

The process involving the use of gaseous hydrocarbons as carbon source has a number of commercial advantages over the process using liquid hydrocarbons. Among these are:

a. Purification of cells is easier than the process involving liquid hydrocarbons which requires a complicated and disadvantageous procedure in separating the cells completely from the remaining hydrocarbons so as to remove an odor of petroleum.

b. There is no unsuitability of the obtained cell cake as foodstuff or feed which is ascribable to the remaining of a tar content containing undesirable impurities such as benzpyrene in the case of using liquid hydrocarbons.

c. Liquefied petroleum gas consisting mainly of propane and butane, or fuel gases discharged from the petroleum refining apparatus, which have heretofore found applications mainly as fuels, are available at low costs.

We have screened many microorganisms isolated from cores soils and oil brines in oil and natural gas fields, cores of stratigraphic drilling wells and swampy muds, and as a result, found those microorganisms which are able to assimilate C C gaseous hydrocarteriological properties shown in Table i.

TABLE 1 DESCRIPTION OF THE STRAIN Scientific name of organism Morphology Motility Gram Reaction Acid-fastness Yeast extract-Malt extract agar colinies Nutrient agar slant Growth PH Growth Temperature ("C.) Optimum Growth Temperature Oxygen Gelatin Liquefaction Milk Reaction Starch Hydrolysis Nitrate Reduction Catalase Production Cellulose Reaction n-Paraffine Utilization Autotrophic Growth Carbon Utilization Carbonhdrate Fermentation GC content of DNA (36) Corynebaclerium fujiokense (No.2-Sl) Rods, straight or curved,

club'shapcd 0.5 to 0.7 by 1.6 to 4.0 microns. In old cultures generally V-forrned shorter rods or cocci.

negative positive negative circular, capitate, smooth, entire,

amorphous, light orange, opaque, glistening abundant growth, filiform, flat,

glistening, smooth, pale reddish orange aerobe negative alkaline, slowly digested negative negative positive none attacked utilize (C -C C C,

no growth Glucose, gluconate, citrate and succinate are utilized as sole carbon sources.

Neither acid nor gas are produced from glycerol, xylose, sucrose, lactose, glucose and starch.

The above given properties were compared with those of known bacteria in accordance with Bergeys, Manual of Determinative Bacteriology, 7th edition, and it was found that the present strain belongs to Corynebacterium but differs from any of the known species in respect of morphology, color and capability of forming acid from saccharose. Hence, it was identified as a new species of the genus Corynebacterium, and named Corynebacterium fujiokense. Strain No. 2-51 was deposited in Fermentation. Research Institute,

Agency of Industrial Science and Technology, Japan under the deposit number FERM-P No. 383, and also deposited in American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under deposit number ATCC 21496.

The new species Nocardia neaopaca used in the present invention has bacteriological properties described in Table 2.

TABLE 2 DESCRIPTION OF THE STRAIN Scientific name of organism Nocardia neoapam (No2-5 3 M orphology Long branching filaments or rods, straight or curved, 0.6 to 0.8 by 2.0 to [4.0 microns. In old cultures generally shorter rods. Aerial mycelium are not observed. Motility negative Gram Reaction positive Acid-fastness negative Yeast extract-Malt extract agar colonies circular, capitate, smooth. entire or filamentous, amorphous, pale pink, opaque, dull abundant growth, tiliform, raised,

Mutrient agar slant glistening, smooth, pale pink none attacked utilize (C -C C -C no growth Glucose, gluconate, citrate and succinate are utilized as sole carbon sources.

Neither acid nor gas are produced from glycerol, xylose, sucrose, lactose, glucose and starch.

Cellulose Reaction n-Paraffine Utilization Autotrophic Growth Carbon Utilization Carbohydrate Fermentation GC content of DNA These properties shown in Table 2 were compared with those of known species in accordance with the classification standards described in Bergeys, Manual of Determinative Bacteriology," 7th edition, and it was found that this strain is similar to Nocardia opaca in respect of morphological properties, color of the slant culture and physiological properties, but differs from the reference strain in that acid-fastness is not observed in any stage of culturing, no aerial hypha is observed, and that the GC base composition of DNA is different. Hence, this strain has been identified as a new species of the genus Nocardia, and named Nocardia Neoopaca. Strain No. 2-53 was deposited in the above-mentioned institution of the Japanese government under the deposit number FERM-P No. 378, and also deposited in American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under deposit number ATCC 21499.

The new species Arthrobacter rubellus used in the present invention has the bacteriological properties shown in Table 3 below. This new species and the new species shown in Table 4 both belong to the genus Arthrobacter, and come next to the species shown in Table l in the order of suitability for use in the present invention.

TABLE 3 DESCRIPTION OF THE STRAIN Scientific name of organism Morphology Motility Gram Reaction Acid-fastness Yeast extract-Malt extract agar colonies circular, pulvinate, rugose, entire,

amorphous, reddish orange, opaque, dull Nutrient agar slant abundant growth, filiform, raised,

dull, rugose, light reddish orange Growth PH 5.5-l0.0

Growth Temperature (C.) -42 Optimum Growth Temperature -37 Oxygen aerobe Gelatin Liquefaction negative Milk Reaction alkaline, slowly digasted Starch Hydrolysis negative Mitrate Reduction negative Catalase Production positive none attacked utilize (C -C no growth Glucose, glucomate citrate and glycerol are utilized as sole carbon sources.

Neither acid nor gas are produced from glycerol, xylose, sucrose, lactose, glucose and starch.

Cellulose Reaction n-Paraffine Utilization Autotrophic Growth Carbon Utilization Carbohydrate Fermentation GC content of DNA The properties shown in Table 3 were compared with those of known species in accordance with the classification standards described in Bergeys, Manual of Determinative Bacteriology, 7th edition, and it was found that this species belongs to the genus Arthlobacter, but differs from the known species in respect of color and physiological properties. Strain No. 2-04 was deposited in the above-mentioned institution of the Japanese government under deposit number FERM-P No. 382, and also deposited in American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., under deposit number ATCC The new species Arthrobacter petroleophagus used in the present invention have the bacteriological properties shown in Table 4 below.

TABLE 4 DESCRIPTION OF THE STRAIN Scientific name of organism Morphology Motility Gram Reaction Acid-fastness Yeast extract-Malt extract agar colonies Nutrient agar slant Growth PH Growth Temperature (C.) Optimum Growth Temperature Oxygen Gelatin Liquefaction Milk Reaction Starch Hydrolysis Nitrate Reduction Catalase Production Cellulose Reaction n-Paraffine Utilization Autotrophic Growth Carbon Utilization Carbohydrate Fermentation GC content of DNA Arthrobacter petroleophagus (No2-l 5) Rods, straight or curved,

club-shaped, 0.4 to 046 by L4 to 1.6 microns. ln old cultures V-formed shorter rods or cocci.

negative positive or negative (positive in old cells) negative circular, capitate, smooth, entire,

amorphous, pale orange, opaque, glistening abundant growth, tiliform,

pulvinate, glistening, smooth, pale yellow orange Strain No. 2-15 was deposited in the abovementioned Japanese government institution under the deposit number FERM-P N0. 38l, and also in American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under deposit number ATCC 21494.

All of the four species of microorganisms employed in the process of the present invention are new ones.

Various microorganisms which belong to the genus Corynebacterium have been known to assimilate liquid hydrocabons, but no report has yet been made heretofore about the species which assimilate gaseous hydrocarbons. As to the microorganisms which belong to the genus Nocardia and assimilate gaseous hydrocarbons, .l. B. Davis, H. H. Chase and R. L. Raymond, Appl. Mikrobioh, 4 (6), 310-315 (1956) describes the presence of species which assimilate ethane (C but the writers are quite silent on the names of such species nor its bacteriological characteristics.

As to the microorganism which belongs to the genus Arthrobacter and assimilates gaseous hydrocarbons, British Pat. No. 1,166,964 published after the priority date of the present application discloses the presence of Arthrobacter roseoparaffinus.

The new species shown in Tables 1 to 4 all assimilate C -C gaseous hydrocarbons, and therefore, a culture medium containing a gaseous C C, hydrocarbon as a carbon source is employed. The culture medium usually contains a nitrogen source source additionally, and preferably minerals.

The term culture medium containing a C -C gaseous hydrocarbon as a carbon source refers not only to a carbon source consisting of one or more of C C, hydrocarbons such as ethane, propane and n-butane, but also to a carbon source consisting of at least 50 percent by weight of one or more of such hydrocarbons and a minor amount of other hydrocarbons such as olefins and isoparaffins. Hence, C fraction remaining after recovering ethylene and butadiene from a cracked product of naphtha, or a gaseous mixture of liquefied petroleum gas consisting predominantly of propane and butane can also be used in the present invention.

Usually, the culture medium used in the present invention contains a nitrogen source besides the carbon source hereinbefore described. As such nitrogen source, we can mention organic nitrogen sources such as urea, pepton, meat-extract, yeast-extract, maltextract, and amino acids and inorganic nitrogen sources such as ammonium chloride, ammonium nitrate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium hydrogenphosphate, ammonium dihydrogenphosphate and sodium nitrite.

it is preferred that the culture medium used in the present invention further contains minerals, examples of which are dipotassium phosphate potassium phosphate, disodium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, ferric chloride, ferrous sulfate and ferric sulfate. Preferably, the culture medium further contains a critical amount of an inorganic salt such as molybdenum oxide, zinc sulfate, copper sulfate, boric acid, manganese sulfate and cobalt chloride. It is sufficient that the amounts of minerals are about 5 X to 5 X 10 percent based on the weight of the medium.

There is no particular limitation on the amount of the gaseous carbon source used in the present invention. But usually the amount recommended ranges 50 500 percent by volume (at 30C.), preferably 100 200 percent by volume, based on the total volume of the culture medium. In a continuous method, either fewer or more amounts can be used as the waste gas can be recycled.

The amounts of the nitrogen source may vary over a considerably wide range, but most commonly 0.02

0.5 percent by weight, preferably0.l 0.2 percent by weight can be used on the basis of the total weight of the culture medium.

Some examples of the culture medium suited for use in the present invention. excepting a carbon source are as follows:

a. Aqueous medium with a pH adjusted to 7.2 which has been prepared. by dissolving in 1 liter of tap water 1 g of NH Cl, 0.5 g of K HPO 0.2 g of MgSO -7H O, 0.01 g of CaCl -2H O, 0.001 g of FeCl -6l-l O, 10 pg of M00 7'0 pgOfZnSO 7H O, 5 pg of CuSO .-5H O, 10 pg of H 30 10 pg of MnSO -5H O and 10 pg of CoCl -6l-l O.

b. Aqueous medium with a pH adjusted tov 7.2 which has been prepared. by dissolving in 1 liter of tap water 1 g of NH Cl, 0.5 g of Na HPO 0.2 g of KH PO,,, 0.2 g of MgSO 7H 0, 0.01 g of CaCl '2- H O, 0.001 g of TeC1 -6H O, 10 pg of M00 pg of ZnSO -7H O, 5 pg of CuSO -SH O, 10 pg of H 10 pg of MnSO SH O and 10 pg of CoCl -6H O.

. Aqueous medium with a pH adjusted to 7.2 which has been preparedby dissolving into one liter of tap water 2 g of NaNo 0.5 g of Na i-IP0 0.2 g of KH PO 0.2 g of MgSO -7H O, 0.01 g of CaCl -2- H O, 0.002 g of FeSO -7H O, 10 pg of M00 70 g of ZnSO '7H O, 5 pg of CuSO -5H O, 10 pg of H 30 10 g of MnSO -5H O and 10 pg of CoC1 -6H O.

Prior to inoculating microorganisms in a culture medium, sterilization of the culture medium is carried out in accordance with a customary method. Most commonly, heat sterilization can be effected at i 10C. for about 15 to 30 minutes. It is a usual practice to inoculate a strain in a heat sterilized culture medium, and introduce a gaseous mixture of a gaseous hydrocarbon and air or oxygen or a gaseous mixture of carbon dioxide, hydrogen and air or oxygen through a sterilizing filter.

1n the present invention using a gaseous carbon source, the employment of pressure culturing leads to an increase of solubility of the carbon source in a culture liquor. In addition, depending upon the degree of pressure,.the gaseous carbon source is readily liquefied, and in the liquid state, is assimilated by microorganisms. This is effective also in inhibiting the generation of heat in a fermentation process, and an increase in the yield of cells can be expected.

The culturing temperation that can be employed in the invention ranges from about 20C. to about 40C., preferably 25 35C., more preferably 30 3C. The pH of the culture medium that can be used in the invention is about 5 to about 10, preferably 6 to 8. Culturing is effected under aerobic conditions, and known aerobic culturing means such as a batchwise shaking culture method and a continuous stirring culture method can be employed in the presence of a molecular oxygencontaining gas, such as oxygen, air or a mixture thereof.

The culturing time may vary depending upon the strain used, composition of the culture medium, carbon source used, culturing temperature, pH of the culture medium or culturing means, but is usually from 3 to 10 days, preferably from 5 to 7 days.

After the end of culturing, cells are separated and recovered from the culture broth, and if desired, subjected to purification.

Example 1 NH Cl (1 g), K HPQ, (0.5 g), MgSO -7H O (0.2 g), CaCl '2H O (0.01 g), FeCl '6H O (0.001 g), MOO 10 pg), ZnSO -7H O (70 #8), CuSO 5H O (5 ,u.g),ll BO (l #8), MnSO.,-H,O Mg) and CoCl -6H O (10 g) were dissolved in 1 liter of tap water and the pH was adjusted to 7.2. 100 ml of the so formed culture medium was put into a 500 ml gas flask and sterilized at 120C. for 20 minutes. Then, one platinum-loopful of Nocardia neoopaca strain No. 2-53 was inoculated into the culture medium and a mixed gas of ethane and air at a volume ratio of 1:3 was introduced into a glass flask through a sterilizing filter. The culturing was conducted at 28C. for 7 days under shaking. Cells growing in the culture medium were centrifugally separated and recovered. The collected cells were washed three times with tap water, and freeze-dried. The gas compositions before and after were determined by gas chromatography analysis. As a result, it was confirmed that the yield of cells was 42 percent by weight based on ethane consumed during culturing and the protein content of the dried cells was 48.3 percent by weight.

Example 2 Nocardia neoopaca strain No. 2-53 was cultured at 28C. for 7 days using the same culture medium and procedure as described in Example 1 except that propane was fed as carbon source. The culture broth was washed three times with tap water and dried in a drier. The yield of cells was 38 percent by weight based on propane consumed during culturing, and the protein content of the dried cells was 40.0 percent by weight. 45

The amino acid composition and vitamin content of the cells are shown-in Table 5. From this table it is seen that the cells have a high nutritive value.

Example 3 Corynebacterium fujiokense strain No. 2-51 was cultured at 30C. for 7 days by using ethane as carbon source using the same culture medium and procedure as described in Example 1. The resulting culture broth was treated in the same manner as in Example 2. The yield of the cells was 41 percent by weight based on ethane consumed during culturing and the protein content of the dried cells was 52.2 percent by weight.

The amino acid composition and vitamin content of the cells are shown in Table 5.

Example 4 Corynebacterium fujiokense strain No. 2-51 was cultured at 30C. using the same culture medium and culturing conditions as described in Example 1 and employing propane or n-butane as carbon source. In the case of n-butane, the n-butane: air volume ratio was adjusted to 1:4. The culture broth was treated in the same manner as in Example 1. When propane was fed as carbon source, the yield of the resulting cells was 39 percent by weight based on consumed propane and the 5 protein content of the dried cells was 50.1 percent by weight. in the case of n-butane, the yield of the resulting cells was 34 percent by weight based on consumed n-butane and the protein content of the dried cells was 48.7 percent by weight.

Example 5 Arthrobacter petroleophagus strain No. 2-15 was cultured at 28C. for 7 days using the same culture me dium and procedure as described in Example 1 and using ethane as carbon source. The culture broth was treated in the same manner as in Example 2. The yield of the cells was 43 percent by weight based on consumed ethane. The protein content of the dried cells was 40.8 percent by weight.

Example 6 By employing propane or n-butane as carbon source, Arthroba'cter rubbelus strain No. 2-04 was cultured for 7 days under the same conditions as adopted in Exampie 2 or 4. The culture broth was treated in the same manner as in Example 1. In the case of propane, the yield of the resulting cells was 38 percent by weight based on consumed propane and the protein content of the dried cells was 41.2 percent by weight. In the case of n-butane, the yield of the resulting cells was 31 percent by weight based on consumed n-butane and the protein content of the dried cells was 45.7 percent by weight. The amino acid composition and vitamin content of the cells produced in the case of n-butane are 35 shown in Table 5.

TABLE 5 WT [Amino Acid Composition and Vitamin Content] Example number 2 3 Nocardia Coryncbac- Artlgzollzacneoo aca teri'um tar ru 2 us g tram fujiokensc, Strain N 0. Strain No. No. Strains 2-53 2-51 2-04.

Carbon source Propane Ethane n-Butane 50 Amino acid composition (weight percent based on proteins):

5. 5. 34 5. 0!] 2. 27 1. 63 1. 39 0. 40 0. 32 0. 22 Non-essential amino acids:

Serinc 3. 23 3. 24 3. 62 Glutamlc acid. 12 06 10.38 11. 02 lrolinm 3. 2i! 3. 74 3.19 Glycine. 4. 20 4. 87 5. 10 Alanine. 8. 52 8.14 8. 4X Tyrosine 2. 2| 2.13 2. 37 Aspartic acid. 7. e0 8. 13 8. 29 Vitamin content (mg/kg):

Vitamin ll; 17 23 1!! Vitamin B 43 40 62 Vitamin B 1153 Vitamin 13 1. 11.0 9.6 11.8 Nlcotinic acid 200 168 Pantothenic acid 2s 31 24 We claim:

1. A process for recovering protein-containing cells which comprises culturing a microorganism selected from the group consisting of species Corynebacterium fujiokense (ATCC 21496), Nocardia neoopaca (ATCC 21499), Arthrobacter rubellus (ATCC 21495) and Arthrabacter petroleophagus (ATCC 21494) under aerobic conditions in a culture medium containing a gaseous C -C hydrocarbon as a carbon source, and separating and recovering cells from the culture broth.

2. The process of claim 1 wherein said culture medium further contains a nitrogen source.

3. The process of claim 2 wherein said culture medium further contains mineral.

4. The process of claim 2 wherein said nitrogen source is selected from the group consisting of ure 2 1 ammonium chloride, ammonium sulfate, sodium nitrate, potassium nitrate, ammonium hydrogenphosphate and ammonium dihydrogenphosphate.

5. The process of claim 3 wherein said mineral is selected from the group consisting of dipotassium phosphate, potassium phosphate, disodium phosphate, so-

dium phosphate, magnesium sulfate, calcium chloride,-

ferric chloride, ferrous sulfate, ferric sulfate, molybdenum oxide, zinc sulfate, copper sulfate, boric acid, manganese sulfate and cobalt chloride.

6. The process of claim 1 wherein the culturing is carried out at a temperature of about 20C. to about 40C. at a pH of about 5 to about 10.

7. The process of claim 6 wherein the culturing tem' perature is 25 C., and the pH of the culture medium is 6 to 8.

8. The process of claim 1 wherein said carbon source contains at least 50 percent by weight of a gas selected from the group consisting of ethane, propane, butane and mixtures thereof. 

2. The process of claim 1 wherein said culture medium further contains a nitrogen source.
 3. The process of claim 2 wherein said culture medium further contains mineral.
 4. The process of claim 2 wherein said nitrogen source is selected from the group consisting of urea, ammonium chloride, ammonium sulfate, sodium nitrate, potassium nitrate, ammonium hydrogenphosphate and ammonium dihydrogenphosphate.
 5. The process of claim 3 wherein said mineral is selected from the group consisting of dipotassium phosphate, potassium phosphate, disodium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, ferric chloride, ferrous sulfate, ferric sulfate, molybdenum oxide, zinc sulfate, copper sulfate, boric acid, manganese sulfate and cobalt chloride.
 6. The process of claim 1 wherein the culturing is carried out at a temperature of about 20*C. to about 40*C. at a pH of about 5 to about
 10. 7. The process of claim 6 wherein the culturing temperature is 25* - 35*C., and the pH of the culture medium is 6 to
 8. 8. The process of claim 1 wherein said carbon source contains at least 50 percent by weight of a gas selected from the group consisting of ethane, propane, butane and mixtures thereof. 